In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.